The objective of the proposed work is to design potent multivalent ligands that influence the differentiation of adult neural stem cells (NSCs) and human pluripotent stem cells (hPSCs) based on a biomimetic strategy - multivalency. Cellular signal transduction can often begin with the multivalent binding of ligands, either secreted or cell-surface tethered, to target cell receptors, leading to receptor clustering. The capacity to control multivalent interactions and thereby modulate key signaling events within living systems is, however, currently very limited. While antibody-induced ligand or receptor clustering has been achieved, this method is not well- controlled, efficient, or readily reproducible. Intracellulr targets can be clustered by the small-molecule dependent dimerization of repeated inducible dimerizing domains, but this approach involves overexpressing fusion proteins and is not readily applicable for endogenous ligands or receptors. The use of synthetic multivalent ligands is a promising approach to control and to elucidate fundamental mechanisms in cellular signaling. If such multivalent ligands could be designed to activate key signaling pathways and thereby control stem cell fate in vitro and in vivo, they could serve as both powerful biological tools and as potent therapeutics. The first aim of the proposed work is to harness multivalent ephrin conjugates to study mechanisms by which Eph-ephrin signaling regulates cell fate decisions in neural stem cells and pluripotent stem cells. Within this aim, we will conduct a structure-function analysis of ephrin multivalency in signaling and stem cell differentiation, as well as engineer peptide-based multivalent ligands for potent activation of Eph-ephrin signaling in vitro and in vivo. Our second aim is to determine whether multivalent conjugates can be engineered to activate Wnt signaling in NSCs, which will be achieved with a combination of engineering peptide-based multivalent ligands and characterizing their signaling properties in vitro and in vivo. We anticipate that the resulting multivalent ephrin and Wnt ligands will serve as potent bioactive materials for controlling stem cell fate decisions, a capability that would be significan for mechanistic investigations in stem cell and developmental biology in vitro and in vivo, as well as for applications including enhanced cell culture systems, pharmacology and toxicology screens, and regenerative medicine approaches to restore organ function.